Auxinic analogues of indole-3-acetic acid

ABSTRACT

The present invention provides compounds and compositions capable of stimulating plant growth, regeneration of plant cells and tissues, and transformation of plant cells and tissues, comprising mono- and multi-substituted auxinic analogues of indole-3-acetic acid (IAA) comprising substituent groups such as halo-, alkyl-, alkoxy-, acyl-, acylamido- and acyloxy-groups. The invention relates to a method of using such mono- and multi-substituted auxinic analogues of IAA to affect growth, regeneration or transformation in monocotyledonous and dicotyledonous plants, as well as in transgenic plant tissues. The invention also contemplates the use of these auxinic IAA analogues in the presence of other plant growth regulators, such as cytokinin, etc., to enhance plant growth.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent applicationSer. No. 08/758,416, filed Nov. 29, 1996 which is a continuation-in-partof provisional application No. 60/007,770 filed Nov. 30, 1995, andcopending application Ser. No. 08/430,209 filed Apr. 27, 1995, all ofwhich are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

[0002] This invention relates to the use of an indole-3-acetic acid(IAA) analogue as a plant hormone stimulatory to plant growth, toregeneration of plant cells and tissues, and to transformation of plantcells. It particularly relates to the use of mono- and multi-substitutedIAA molecules. The invention also relates to compositions comprising IAAanalogues of the invention.

BACKGROUND OF THE INVENTION

[0003] Plant growth is affected by a variety of physical and chemicalfactors. Physical factors include available light, day length, moistureand temperature. Chemical factors include minerals, nitrates, cofactors,nutrient substances and plant growth regulators or hormones, forexample, auxins, cytokinins and gibberellins.

[0004] Indole-3-acetic acid (IAA) is a naturally-occurring plant growthhormone identified in plants. IAA has been shown to be directlyresponsible for the increase in growth in plants in vivo and in vitro.The characteristics influenced by IAA include cell elongation,internodal distance (height), leaf surface area and crop yield. IAA andother compounds exhibiting hormonal regulatory activity similar to thatof IAA are included in a class of plant regulators called “auxins.”

[0005] Preparations based on cytokinins, such as 6-furfurylamino purine(kinetin) and 6-benzylamino purine (BAP), are also known to be growthstimulators. However, cytokinin-based preparations are most effective incombination with auxins. While the mechanism by which cytokinins affectthe growth cycle of plants is far from being understood, it is apparentthat they affect leaf growth and prevent aging in certain plants.

[0006] It is a general objective in the field to successfully engineerand regenerate plants of major crop varieties using methods such astissue culture and genetic engineering. Major crop varieties ofparticular interest in this regard are agricultural crops such as maize,wheat, rice, soybeans and cotton.

[0007] To regenerate plants, in vitro culture techniques have beenestablished. (Reinert, J., and Bajaj, Y. P. S., eds. (1977) Plant Cell,Tissue and Organ Culture, Berlin: Springer; Simmonds, N. W. (1979)Principles of Crop Improvement, London: Longman; Vasil, I. K., Ahuja, M.K. and Vasil, V. (1979) “Plant tissue cultures in genetics and plantbreeding,” Adv. Genet. 20:127-215.) Specific in vitro culture techniquesto regenerate plants include embryo culturing shoot tip culturing andcallus, cell and protoplast culturing. Embryo culturing has been shownto be important in making difficult interspecies crosses, whileshoot-tip culturing is important in rapid clonal multiplication,development of virus-free clones and genetic resource conservation work.Callus, cell, and protoplast cultures have been shown to be importantfor cultures in which organization is lost but can be recovered.

[0008] Plant genetic engineering techniques have also been established.These techniques include gene transfer by transformation or byprotoplast fusion. In gene transfer, the steps involved are: (a)identification of a specific gene; (b) isolation and cloning of thegene; (c) transfer of the gene to recipient plant host cells: (d)integration, transcription and translation of the DNA in the recipientcells; and (e) multiplication and use of the transgenic plant (T.Kosuge, C. P. Meredith and A. Hollaender, eds (1983) Genetic Engineeringof Plants, 26:5-25; Rogers et al. (1988) Methods for Plant MolecularBiology [A. Weissbach and H. Weissbach, eds.] Academic Press, Inc., SanDiego, Calif.). In protoplast fusion, plant cell protoplasts are fusedby standard chemical (e.g., PEG) or electroporation techniques. Afterregeneration of the fused cells, interspecies amphidiploids have beenobtained. The technique may provide desired amphidiploids which cannotbe made by conventional means, and presents possibilities for somaticrecombination by some variant of it. The foregoing techniques are widelyin use (Chaleff, R. S. (1981) Genetics of Higher Plants, Applications ofCell Culture, Cambridge: Cambridge University Press), and newly insertedforeign genes have been shown to be stably maintained during plantregeneration and are transmitted to progeny as typical Mendelian traits(Horsch et al. (1984) Science 223:496, and DeBlock et al. (1984) EMBO3:1681). These foreign genes retain their normal tissue specific anddevelopmental expression patterns.

[0009] The Agrobacterium tumefaciens-mediated transformation system hasproved to be efficient for many dicotyledonous plant species. Forexample, Barton et al. (1983, Cell 32:1033) reported the transformationand regeneration of tobacco plants, and Chang et al. (1994, Planta5:551-558) described stable genetic transformation of Arabidopsisthaliana.

[0010] The Agrobacterium method for gene transfer was also applied tomonocotyledonous plants, e.g., in plants in the Liliaceae andAmaryllidaceae families (Hooykaas-Van Slogteren et al., 1984, Nature311:763-764) and in Dioscorea bulbifera (yam) (Schafer et al., 1987,Nature 327:529-532); however, this method did not appear to be efficientfor the transformation of graminaceous monocots, which include such foodcrops as wheat, rice and corn.

[0011] Transformation of food crops was obtained with alternativemethods, e.g., by polyethylene glycol (PEG)-facilitated DNA uptake(Uchimiya et al. (1986) Mol. Gen. Genet. 204:204-207) andelectroporation (Fromm et al. (1986) Nature 319:791-793), both of whichused protoplasts as transfer targets. Monocot and dicot tissues may betransformed by bombardment of tissues by DNA-coated particles (Wang etal. (1988) Plant Mol. Biol. 11:433-439; Wu, in Plant Biotechnology(1989), Kung and Arntzen, Eds., Butterworth Publishers, Stoneham,Mass.). Regeneration was described in rice (Abdullah et al. (1986)Bio/Technology 4:1087-1090) and maize (Rhodes et al. (1988)Bio/Technology 6:56-60 and (1988) Science 240:204-207).

[0012] Thus, although regeneration and transformation protocols havebeen established, there remains a need to stimulate regeneration andtransformation of monocotyledonous and dicotyledonous plants. Indeed,some plants have been difficult to regenerate and transform [Vasil andVasil (1994) in Plant Cell and Tissue Culture (Vasil and Thorpe, eds.),Kluwer Academic Publishers, Dordrech, Netherlands; Chee (1995) PlantCell Reports 14:753-757; Bums and Schwartz (1996) Plant Cell Reports15:405-408; Mihaljevic et al. (1996) Plant Cell Reports 15:610-614;Schopke et al. (1996) Nature Biotechnology 14:731). Moreover, there is aneed to stimulate growth of the plants, particularly aftertransformation and regeneration.

SUMMARY OF THE INVENTION

[0013] The present invention satisfies these needs by providingcompounds and compositions 0.10 which stimulate plant growth,regeneration of plant cells and tissues, and transformation of plantcells and tissues. The compounds of the invention comprise mono- ormulti-substituted IAA (indole-3-acetic acid) or ester or saltderivatives thereof. The invention also provides compositions comprisingone or more of these IAA analogues and, optionally, a carrier. Theinvention contemplates the use of such auxinic analogues to affectgrowth, regeneration and transformation in both monocotyledonous anddicotyledonous plants. In particular, the invention providesmonosubstituted IAA analogues having a substituent group at the 2, 4, 5,6 or 7 position of the IAA structure, wherein said substituents arehalo- or alkyl-, alkoxy-, acyl-, acylamido- and acyloxy-substituentgroups having 1-10 carbon atoms. The invention also providesmulti-substituted IAA analogues having two to five, same or different,substituent groups at different positions selected from positions 2, 4,5, 6 or 7 of the IAA structure wherein said substituents are halo- oralkyl-, alkoxy-, acyl-, acylamido- and acyloxy-substituent groups having1-10 carbon atoms.

[0014] The compositions of the invention may include, in addition to oneor more of the mono- or multi-substituted compounds, one or moreadditional plant growth regulators. Such plant growth regulatorsinclude, for example, a cytokinin, a gibberellin, etc., in definiteproportions for wide application to various plants. In specificembodiments, the invention is exemplified with compositions comprisingmono- or multi-substituted IAA analogues having between one and five,same or different, substituent groups that are halo-, alkyl-, alkoxy-,acyl-, acylamido- or acyloxy-substituent groups at positions 2, 4, 5, 6and/or 7 of the IAA structure, and a cytokinin to affect the growth ofplants.

[0015] The invention further relates to media comprising the compoundsand compositions of the invention. Such media comprise one or more IAAanalogues and optionally an IAA analogue and optionally a plant growthregulator, e.g., cytokinin, to stimulate plant growth, to stimulateregeneration of plant cells and tissues, and to stimulate transformationof plant cells and tissues.

[0016] The invention also relates to a method of stimulating plantgrowth comprising (a) applying to a plant, plant cell or tissue aneffective amount of the compound or composition of the invention andoptionally, applying one or more additional plant growth regulators, forexample, a cytokinin, a gibberellin, etc., and (b) incubating the plantcell or tissue under conditions sufficient to stimulate the regenerationof the plant cell or tissue.

[0017] The invention also provides a method for stimulating theregeneration of plant cells and/or tissues comprising (a) applying to aplant cell or tissue an effective amount of the compound or compositionof the invention and applying one or more additional plant growthregulators, for example, a cytokinin, a gibberellin, etc., and (b)incubating the plant cell or tissue under conditions sufficient tostimulate the regeneration of the plant cell or tissue.

[0018] Also, the invention provides a method for stimulating thetransformation of plant cells and/or tissues comprising (a) contactingthe plant cell or tissue with a nucleic acid molecule (e.g., bytransformation or protoplast fusion), (b) applying to the plant cell ortissue an effective amount of a compound or composition of the inventionand, optionally, one or more additional plant growth regulators, forexample, a cytokinin, a gibberellin, etc., and (c) incubating the plantcell or tissue under conditions sufficient to stimulate transformationof the plant cell or tissue with the nucleic acid molecule. Thecompounds and compositions of the invention also may be used tostimulate regeneration or growth of the transformed tissue or cells,thus providing a method to obtain a transgenic plant.

[0019] The invention also concerns a method of attenutating oralleviating environmental stress in a plant, plant cell or tissuecomprising (a) contacting a plant, plant cell or tissue which has beenexposed to an environmental stress (such as drought, excess temperature,diminished temperature, chemical toxicity [e.g., antibiotic,herbicides], pollution, excess light, and diminished light) with aneffective amount of the compounds or compositions of the invention, and(b) incubating said plant, plant cell or tissue under conditionssufficient to attenuate or alleviate said stress.

DESCRIPTION OF THE FIGURES

[0020]FIG. 1 presents the chemical structure of IAA where R₁-R₅ arehydrogen and the numbers (1)-(7) represent the numbering pattern for theIAA chemical structure.

[0021]FIG. 2 presents the chemical structures of some halogenated IAAauxinic analogues.

[0022]FIG. 3 presents the chemical structures of some mono-substituted,alkyl-IAA auxinic analogues having an alkyl group in the 4 position. Thepresent invention also contemplates alkyl-IAA compounds having the samealkyl substituent group at position 2, 5, 6 or 7. Exemplified in FIG. 3are alkyl-IAA structures having an alkyl (R) group with 1-4 carbonatoms. The instant invention, however, provides IAA analogues with alkylsubstituents with 1-10 carbon atoms.

[0023]FIG. 4 presents the chemical structures of some mono-substituted,alkoxy-IAA auxinic analogues having an alkoxy group in the 4 position.The present invention also contemplates alkoxy-IAA compounds having thesame alkoxy substituent group at position 2, 5, 6 or 7. Exemplified inFIG. 4 are alkoxy-IAA structures having an alkoxy group with 1-4 carbonatoms. The instant invention, however, provides IAA molecules withalkoxy substituents with 1-10 carbon atoms.

[0024]FIG. 5 presents the chemical structures of some mono-substituted,acyl-IAA auxinic analogues having an acyl group in the 4 position. Thepresent invention also contemplates acyl-IAA compounds having the sameacyl substituent group at position 2, 5, 6 or 7. Exemplified in FIG. 5are acyl-IAA structures having an acyl group with 1-4 carbon atoms. Theinstant invention, however, provides acyl-substituted IAA moleculeshaving acyl groups with 1-10 carbon atoms.

[0025]FIG. 6 presents the chemical structures of some mono-substituted,acylamido-IAA auxinic analogues having an acylamido group in the 4position. The present invention also contemplates acylamido-IAAcompounds having the same acylamido substituent group at position 2, 5,6 or 7. Exemplified in FIG. 6 are acylamido-IAA structures having anacylamido group with 1-4 carbon atoms. The instant invention, however,provides acylamido-substituted IAA molecules having acylamide groupswith 1-10 carbon atoms.

[0026]FIG. 7 presents the chemical structures of some mono-substituted,acyloxy-IAA auxinic analogues having an acyloxy group in the 4 position.The present invention also contemplates acyloxy-IAA compounds having thesame acyloxy substituent group at position 2, 5, 6 or 7. Exemplified inFIG. 7 are acyloxy-IAA structures having an acyloxy group with 1-4carbon atoms. The instant invention, however, provides acyloxysubstituted IAA molecules having acyloxy groups with 1-10 carbon atoms.

[0027]FIG. 8 documents tomato plant growth with increasingconcentrations of 6-BrIAA (0.5, 2.0 and 8.0 mg/l) in the absence (toprow) and in the presence (bottom row) of BAP (0.5 mg/l) on the formationof roots (top row) and calli (bottom row).

[0028]FIG. 9 documents potato plant growth with increasingconcentrations of 6-BrIAA (0.5, 2.0 and 8.0 mg/l) in the absence (toprow) and in the presence (bottom row) of BAP (0.5 mg/l) on the formationof roots (top row) and calli (bottom row).

[0029]FIG. 10 documents tobacco plant growth with increasingconcentrations of 6-BrIAA (0.5, 2.0 and 8.0 mg/l) in the absence (toprow) and in the presence (bottom row) of BAP (0.5 mg/l) on the formationof roots and calli (top row) and shoots and calli (bottom row).

[0030]FIG. 11 documents cassava plant growth in the presence of 5-FIAAor 7-FIAA (2.0 mg/l) and BAP (0.5 mg/l) on shoot formation.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The following definitions are provided in order to provideclarity as to the intent or scope of their usage in the specificationand claims.

[0032] The term indole-3-acetic acid or IAA as used herein refers to thechemical structure of FIG. 1 where R₁-R₅=hydrogen. This term refers notonly to the free acid form but also to an amide, an ester or a salt formof IAA. Included in the meaning of IAA are, for example, such salt andester derivatives as the sodium, potassium, ammonium, dimethylamine,ethanolamine, etc. salts and amides and the lower alkyl esters.

[0033] The term monosubstituted IAA as used herein refers to an IAAmolecule of FIG. 1 where one or the R₁-R₅ groups represents a halo-, analkyl-, an alkoxy-, an acyl-, an acylamido- or an acyloxy-substituentgroup at the 2, 4, 5, 6 or 7 position in the IAA chemical structure.

[0034] The term multi-substituted IAA as used herein refers to an IAAmolecule of FIG. 1 where two or more of the R₁-R₅ groups represent thesame or different halo-, alkyl-, alkoxy-, acyl-, acylamido- oracyloxy-substituent group in at least two of the positions correspondingto the 2, 4, 5, 6 or 7 position in the IAA chemical structure.

[0035] The term auxinic analogue(s) of IAA or IAA analogue or IAAauxinic analogue as used herein refers to a mono- or multi-substitutedIAA that comprises, for example, one or more of the groups including ahalo-, an alkyl-, an alkoxy-, an acyl-, an acylamido-, an acyloxy- andthe like.

[0036] As used herein, the analogues include not only the free acid formbut also an amide, an ester or a salt form of the mono- ormulti-substituted IAA analogues.

[0037] A halo-group refers to a halogen including, but not limited to,iodo-, bromo-, chloro- and fluoro-groups.

[0038] An alkyl-group includes, but is not limited to, an alkyl, R—,(linear, branched or cyclic; saturated or unsaturated), wherein R has1-10 carbon atoms.

[0039] An alkoxy-group includes, but is not limited to, an alkoxy, R—O—(linear, branched or cyclic; saturated or unsaturated), wherein R has1-10 carbon atoms.

[0040] An acyl-group includes, but is not limited to, an acyl, R—C(O)—(linear, branched or cyclic; saturated or unsaturated), wherein R has1-10 carbon atoms.

[0041] An acylamido-group includes, but is not limited to, an acylamido,R—C(O)—NH— (linear, branched or cyclic; saturated or unsaturated),wherein R has 1-10 carbon atoms.

[0042] An acyloxy-group includes, but is not limited to, an acyloxy,R—C(O)—O— (linear, branched or cyclic; saturated or unsaturated),wherein R has 1-10 carbon atoms.

[0043] The term plant growth regulator or hormone as used herein refersto a naturally occurring or synthetic compound that acts as a hormone inregulating plant growth. Plant growth regulators are exemplified byauxins, cytokinins and gibberellins.

[0044] The term auxin or cytokinin as used herein refers to a plantgrowth regulator that affects the growth of plants. An auxin isexemplified by a compound such as indole-3-acetic acid (IAA),indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (2,4-D),naphthaleneacetic acid (NAA), 5,6-dichloroindole-3-acetic acid(5,6-Cl₂-IAA) and the like. A cytokinin is exemplified by a compoundsuch as 6-benzylamino purine (BAP), N6 (A₂ isopentenyl) adenine (2iP),isopentenylpyrophosphate (ipp),6-(4-hydroxy-3-methyl-2-transbetenylamino)purine (zeatin),6-furfurylaminopurine (kinetin) and the like. A compound can be testedfor auxin activity using a bioassay, e.g., the elongation of coleoptilesof Avena sativa (Bottger et al. (1978) Planta 140:89) or the root growthinhibition of Chinese cabbage (Marumo et al. (1974) in Plant GrowthSubstance, p. 419, Hirokawa Publishing Co., Inc., Tokyo) or thehypocotyl swelling of mung bean (Marumo et al. (1974) supra). Cytokininactivity may be measured in assays designed to evaluate the promotion ofgrowth in plants (e.g., tobacco bioassays, etc.) as is well known in theart (Skoog et al. 1967) Phytochem 6:1169-1192; Morris (1986) Ann. Rev.Plant Physiol. 37:509-538; Horgan (1984) in Advanced Plant Physiol(Wilkins, M. B., ed.) pp. 53-75, Pitman Publishing, London; Letham andPalni (1983) Ann. Rev. Plant Physiol 34:163-197; and Chen (1981) inMetabolism and Molecular Activities of Cytokinins (Guern, J. andPeaud-Lenoel, C., eds., Springer, New York, pp. 34-43). Variations ofthe cytokinin/auxin concentration ratio cause the enhancement in plantgrowth to occur preferentially in certain tissues. For example, a highcytokinin/auxin ratio promotes growth of shoots, whereas a low cytokininto auxin ratio promotes the growth of roots (Depicker et al. (1983) inGenetic Engineering of Plants, T. Kosunge, C. P. Meredith and A.Hollaender, eds., Plenum Press, New York, p. 154).

[0045] The term medium or media as used herein refers to a solid orliquid comprising nutrient sufficient to support plant cell growth, theregeneration of plant cells and tissues, and the transformation of plantcells and tissues.

[0046] The term carrier as used herein refers to a chemically- orbiologically- or physiologically-acceptable molecule that is hydrophobicof hydrophilic or amphoteric and that is useful in facilitating theeffectiveness of an active ingredient (i.e., an IAA analogue of theinvention) in a plant.

[0047] The term a plant as used herein refers to a whole plant or a partof a plant comprising, for example, a locus of a plant, a cell of aplant, a tissue of a plant, an explant, or seeds of a plant. This termfurther contemplates a plant in the form of a suspension culture or atissue culture including, but not limited to, a culture of calli,protoplasts, embryos, organs, organelles, etc.

[0048] The term transformed plant or transformed plant tissues as usedherein refers to introduction of a nucleic acid molecule, e.g., nativeof foreign DNA, into a plant or plant tissue by transformation orprotoplast fusion.

[0049] The term transgenic plant or transgenic plant tissue as usedherein refers to a plant or plant tissue stably transformed with aforeign gene.

[0050] The term transient expression refers to a plant or plant tissuetransformed with a DNA, where that DNA is expressed only for a shortperiod of time immediately after transformation.

[0051] The term genetic engineering as used herein refers to theintroduction of foreign, often chimeric, genes into one or more plantcells which can be regenerated into whole, sexually competent, viableplants which can be self-pollinated or cross-pollinated with otherplants of the same species so that the foreign gene, carried in the germline, can be inserted into or bred into agriculturally useful plantvarieties.

[0052] The term regeneration as used herein refers to the production ofat least one newly developed or regenerated plant tissue, e.g., root,shoot, callus, etc., from a cultured plant tissue or unit, e.g., leafdisc, seed, etc.

[0053] The terms percent regeneration, % regeneration or regenerationefficiency as used herein refer to the number of tissue cultured plantunits producing at least one newly developed or regenerated tissue as apercentage of the total number of tissue cultured plant units, e.g.,$\left( {\frac{{number}\quad {of}\quad {leaf}\quad {discs}\quad {with}\quad {shoots}}{{total}\quad {number}\quad {of}\quad {leaf}\quad {discs}} \times 100} \right).$

[0054] The terms affecting plant growth or growth affecting or affectoror affect as used herein refer to any one of a number of plant responseswhich improve or change, relative to what is observed in the absence ofthe growth regulator, some characteristic of overall plant growth, forexample, stimulation of seed germination, inducing rooting, suppressingshooting, promoting cell proliferation, stimulating callus growth, etc.

[0055] The term effective amount as used herein refers to the amount orconcentration of a compound that is a plant growth regulator or hormoneadministered to a plant such that the compound stimulates or invokes oneor more of a variety of plant growth responses. A plant growth responseincludes, among others, the induction of stem elongation, the promotionof root formation, the stimulation of callus formation, enhancement ofleaf growth, stimulation of seed germination, increase in the dry weightcontent of a number of plants and plant parts, and the like.

[0056] The present invention relates to the discovery that mono andmulti-substituted IAA analogues have utility as auxins in affectingplant growth. For example, in combining 5-bromo-IAA with cytokinin, bothcallus and shoot formation are observed [disclosed in copending U.S.application Ser. No. 08/430,209 filed Apr. 27, 1995].

[0057] IAA auxinic analogues are compared to IAA in functioning as anauxin in both monocots and dicots. For example, it was found that5-bromo-IAA was between two and four times more effective than IAA instimulating the regeneration of green calli from Arabidopsis thaliana.

[0058] Growth affecting compositions of the present invention comprisean IAA analogue, or a mixture of an IAA analogue and one or moreadditional plant growth regulators, such as cytokinin, gibberellin orthe like, mixed with a carrier or auxiliary nutrients. The use of BAP,2iP and kinetin with an IAA analogue is also exemplified in particularembodiments of this invention. It is contemplated that other cytokininsor other plant growth regulators known to the art can be utilized withan IAA analogue to make a growth affecting composition of the invention.It is also contemplated that more than one cytokinin or a differentplant growth regulator (e.g., gibberellin, etc.) can be admixed with anIAA analogue to make a growth enhancing composition of the invention.Also, the choice of plant growth regulator can be varied at differentstages of the incubation or application cycles characterizing the growthof a particular plant. Plant growth regulators are known to the art andinclude, but are not limited to, BAP, 2iP, ipp, zeatin, kinetin,gibberellin, and the like, as described in Skoog et al. (1967)Phytochemistry 6:1169-1192 and Theologis (1989) in Plant Biotechnology(Kung and Arntzen, eds.) Butterworth Publishers, Stoneham, Mass.

[0059] The mechanism by which the compounds and compositions of thepresent invention affect the growth cycle of plants and plant tissues isnot fully understood at present but it is apparent, as will bedemonstrated hereinafter, that they play a significant role in inducinga number of growth affecting responses in a variety of plant species.

[0060] In particular embodiments of the invention, various IAA analogueswere screened for auxinic activity by incubating different planttissues, e.g., tobacco and tomato leaf discs and potato stems in (a) MScomplete medium (obtained from Life Technologies, Inc., Gaithersburg,Md.) containing different concentrations of auxin only and (2) the MScomplete auxin medium containing different ratios of cytokinin/auxin.

[0061] In a specific embodiment, tomato leaf discs, tobacco leaf discsand potato stems were incubated in the MS complete medium comprisingdifferent amounts (i.e., 0.5, 2.0 and 8.0 mg/l) of 6-BrIAA in theabsence and presence of a cytokinin, e.g., BAP (0.5 mg/l). For tomatoleaf discs, when only 6-BrIAA was present in the incubation mixture,only root formation was observed from the tomato leaf discs. FIG. 9,upper row, shows the direct correlation between root formation fromtomato leaf discs and increasing concentration of 6-BrIAA. However, asshown in FIG. 9, lower row, when the tomato leaf discs were incubated inthe MS complete medium containing 0.5 mg/l BAP and 0.5, 2.0 or 8.0 mg/mlof 6-BrIAA, calli, not roots, were formed in each case.

[0062] When the same auxin and cytokinin conditions were applied topotato stems (FIG. 8) and tobacco leaf discs (FIG. 10), different growthresponses were observed. In potato, root formation increased as theconcentration of 6-BrIAA increased from 0.5 to 2.0 mg/l, but at aconcentration of 8.0 mg/ml, root formation was diminished (FIG. 8, upperrow). When BAP (0.5 mg/l) was present in the incubation mixture, callusformation was observed at all ratios of cytokinin/auxin (FIG. 9, lowerrow). In contrast, when tobacco leaf discs were exposed to differentconcentrations of 6-BrIAA in the absence of BAP (FIG. 10, upper row),both the formation of roots and the formation of calli were observed,while in the presence of both 6-BrIAA and BAP (FIG. 10, lower row), theformation of both shoots and calli occurred. These results indicate thatdifferent plant tissues have different responses to the auxin treatment,and that the concentration of auxin influenced the regeneration of planttissues both qualitatively and quantitatively.

[0063] In another embodiment of the invention, IAA analogues werescreened for auxinic activity. IAA derivative structures such as2-bromoindole-3-acetic acid (2-BrIAA), 6-bromoindole-3-acetic acid(6-BrIAA), 7-bromoindole-3-acetic acid (7-BrIAA),5-chloroindole-3-acetic acid (5-ClIAA), 5-fluoroindole-3-acetic acid(5-FIAA), 7-fluoroindole-3-acetic acid (7-FIAA), 5-iodoindole-3-aceticacid (5-IIAA), 5-ethylindole-3-acetic acid (5-EtIAA),7-ethylindole-3-acetic acid (7-EtIAA), and 5-methoxyindole-3-acetic acid(5-MeOIAA) were tested for the ability to function as an auxin in theregeneration of plant tissues in comparison to auxin standards in theart such as IAA and NAA. Table 1 documents the ability of the differentIAA analogues to stimulate the regeneration of roots or calli fromtobacco, tomato and potato. In general, IAA analogues having a brominesubstitution at the 2 TABLE 1 Regeneration of roots or calli fromdifferent plant tissues using IAA analogues Tobacco Tomato Potato Auxins2 mg/l Roots Callus Roots Callus Roots Callus NAA + + +++ − + +++ IAA++++ + +++ − +++ ++ 2-BrIAA ++++ ++ + − +++ +++ 6-BrIAA +++ +++ ++++ +++++ ++ 7-BrIAA ++++ ++ ++++ − +++ + 5-CIIAA +++ ++ + + − ++++ 5-FIAA++++ + +++ − ++++ + 7-FIAA ++++ +++ ++ + NA NA 5-IIAA + +(−) ++ − +(−) −5-EtIAA + +(−) ++ − ++ − 7-EtIAA − + − − ++++ − 5-MeoIAA ++ ++ − + ++ ++

[0064] or 6 or 7 position or a fluorine substitution at the 5 or 7position appear to exhibit auxinic activity that was equal to or betterthan that observed for IAA.

[0065] These IAA derivatives were further evaluated for auxinic activityin the presence of a cytokinin. Table 2 indicates the ability of thedifferent IAA analogues (at a concentration of 2 mg/l) in the presenceof BAP (at a concentration of 0.5 mg/l) to stimulate the regeneration ofshoots and/or calli from tobacco, tomato and potato. Incubation of planttissues TABLE 2 Regeneration of shoots or calli from different planttissues using IAA analogues together with cytokinin BAP 0.5 mg/l +Auxins Tobacco Tomato Potato 2 mg/l Callus Shoots Callus Shoots CallusShoots NAA ++++ − ++++ − +++ − IAA + ++++ +++ + ++ − 2-BrIAA ++ ++ + −++ − 6-BrIAA +++ + ++++ − +++ − 7-BrIAA + ++++ ++++ − + − 5-CIIAA +++ −+++ + ++ − 5-FIAA +++ ++ ++++ + + − 7-FIAA ++++ ++ ++++ − NA NA 5-IIAA+(−) +++ + − +(−) − 5-EtIAA − +++ +++ − + − 7-EtIAA − ++++ +++ − + −5-MeOIAA − ++++ ++ − ++ −

[0066] in the presence of both auxin and cytokinin promoted callusformation in all three plants tested, i.e., tobacco, tomato and potato,whereas shoots were produced only in tobacco and not in tomato andpotato incubated with both auxin and cytokinin. In general, thehalogenated IAA analogues were comparable to IAA in inducing theregeneration of calli in tobacco, while alkyl-IAA and alkoxy-IAAanalogues showed auxinic activity similar to that of IAA in tomato andpotato under these conditions.

[0067] These IAA analogues were further evaluated for their abilities toeffect the regeneration of plant tissues comprising foreign DNA. Tobaccoand potato tissues were subjected to Agrobacterium-mediatedtransformation techniques, including cocultivation with Agrobacteriumcontaining pBI121 and selecting with MS complete medium containing 0.5mg/l BAP, 2 mg/l IAA derivative and 100 mg/l kanomycin. Table 3indicates that the IAA analogues tested (7-BrIAA, 5-ClIAA, 5-FIAA,7-FIAA, 5-EtIAA, 7-EtIAA and 5-MeOIAA), in the presence of BAP,stimulated the regeneration of kanamycin resistant calli in transformedtobacco and potato, and shoots in transformed tobacco. In general,halogenated-IAA, alkyl-IAA and alkoxy-IAA derivatives exhibited IAA-typeauxinic activities in transgenic plant tissues.

[0068] Further, examples of IAA analogues of the invention wereevaluated for auxinic activity in the presence of a cytokinin. Table 4indicates the ability of 2-BrIAA, 6-BrIAA, 7-BrIAA, 5-FIAA, 5-EtIAA and7-EtIAA (at a concentration of 2 mg/l) in the presence of 0.5 mg/l ofBAP to stimulate the regeneration of shoots and callus from cassavaleaves and stems. It was also shown that 5-FIAA promoted the formationof shoots in cassava stems and to a greater extent from cassava leaves.

[0069] In another embodiment of the invention, IAA analogues of thepresent invention were applied to the regeneration of plant tissuesknown in the art to be difficult to regenerate, such as cassava, woodyplants, and monocotyledonous crops [Vasil and Vasil (1994) in Plant Celland Tissue Culture (Vasil and Thorpe, eds.), Kluwer Academic Publishers,Dordrech, Netherlands; Chee (1995) Plant Cell Reports 14:753-757; Bumsand Schwartz (1996) Plant Cell Reports 15:405-408; Mihaljevic et al.(1996) Plant Cell Reports 15:610-614; Schopke et al. (1996) NatureBiotechnology 14:731]. FIG. 11 documents the regeneration of shoots incassava leaves and stems incubated in MS complete medium comprising 2mg/l of 5-FIAA and 0.5 mg/l BAP. TABLE 3 Regeneration ofAgrobacterium-mediated transformed plant tissues using IAA analoguesTobacco Potato BAP 0.5 mg/+ Callus Shoots Callus Shoots NAA ++ + ++ −IAA ++ + + − 7-BrIAA ++ + +(−) − 5-CIIAA +++ ++ ++ − 5-FIAA +++ ++ + −7-FIAA +++ + +(−) − 5-EtIAA + ++ + − 7-EtIAA + ++ +(−) − 5-MeoIAA ++++ + −

[0070] TABLE 4 Regeneration of shoots or calli from Cassava Leaves andStems with IAA Analogues in the Presence of Cytokinin BAP 0.5 mg/+ StemsLeaves Auxin 2 mg/l Callus Shoots Callus Shoots NAA +++ − +++ − 2-BrIAA++ − + − 6-BrIAA +++ − NT NT 7-BrIAA ++ − +++ − 5-FIAA +++ + ++ +++5-EtIAA ++ − + − 7-EtIAA + − NT NT

[0071] Traditionally, in order to achieve shoot regeneration fromcassava leaves and stems, the cassava tissue is transferred from amedium containing a high amount of auxin to another medium containingauxin and cytokinin. In the present invention, the regeneration ofshoots from cassava tissue was obtained without tissue transfer from ahigh auxin medium to a medium containing auxin and cytokinin. Inaddition, the regeneration of shoots in cassava tissue, according to theinvention, showed significant improvement in the number of shootsregenerated. Thus, the IAA analogues of the present invention not onlyexhibit auxinic activity but also improve the yield of plant tissueregenerated, as exemplified in plant tissues traditionally described asbeing difficult to regenerate, e.g., cassava, woody plants, maize,soybean, wheat, etc.

[0072] The practice of the present invention contemplates a wide varietyof plant growth responses, including stimulation of seed germination andbreaking of dormancy; increasing yields; hastening ripening and colorproduction in fruit; increasing flowering and fruiting; stimulatingshoot formation; inducing callus development; inducing rooting andcausing cell proliferation; increasing the hardiness of various plantspecies; and increasing the dry weight content of a number of plants andplant parts. In addition to these categories of responses, any othermodification of a plant, seed, fruit or vegetable, so long as the netresult is to increase the growth or maximize any beneficial or desiredproperty of the agricultural and horticultural crop or seed, is intendedto be included within the scope of advantageous responses achieved bythe practice of the present invention.

[0073] Suitable applications of the growth enhancing compositions of thepresent invention to cultures of plant tissues induce the regenerationof shoots, roots or calli. This effect occurs in both monocotyledonousand dicotyledonous plant species and applies to a wide variety ofplants.

[0074] The compositions of the instant invention are further utilizedfor plant regeneration from transgenic plants.

[0075] Genetic engineering of plants generally involves twocomplementary processes. The first process involves the genetictransformation of one or more plant cells of a specificallycharacterized type. By transformation it is meant that a foreign gene,typically a chimeric gene construct, is introduced into the genome ofthe individual plant cells, typically through the aid of a vector whichhas the ability to transfer the gene of interest into the genome of theplant cells in culture. The second process then involves theregeneration of the transformed plant cells into whole sexuallycompetent plants. Neither the transformation nor regeneration processneed be 100% successful but must have a reasonable degree of reliabilityand reproducibility so that a reasonable percentage of the cells can betransformed and regenerated into whole plants.

[0076] The two processes, transformation and regeneration, must becomplementary. The complementarity of the two processes must be suchthat the tissues which are successfully genetically transformed by thetransformation process must be of a type and character, and must be insufficient health, competency and vitality, so that they can besuccessfully regenerated into whole plants.

[0077] Successful transformation and regeneration techniques have beendemonstrated for monocots and dicots in the prior art. For example, thetransformation and regeneration of tobacco plants was reported in Bartonet al., Cell 32:1033 (April 1983), whereas the regeneration of cotton isdescribed in Umbeck, U.S. Pat. No. 5,004,863, issued Apr. 2, 1991.Further, transformation and regeneration of rice was described byAbdullah et al. (1986) Bio/Technology 4:1087-1090, whereas maize wastransformed and regenerated as described in Rhodes et al. (1988)Bio/Technology 6:56-60 and Science 240:204-207.

[0078] The most common methodology used for the transformation of cellsof dicot plant species involves the use of the plant pathogenAgrobacterium tumefaciens. Although Agrobacterium-mediatedtransformation has been achieved in some monocots, other methods of genetransfer have been more effective, e.g., the polyethylene glycol method,electroporation, direct injection, particle bombardment, etc., asdescribed by Wu in Plant Biotechnology (1989) pp. 35-51, ButterworthPublishers, Stoneham, Mass. The present invention will be useful withany method of transformation that includes plant regeneration steps.

[0079] In a specific embodiment, the invention envisions the genetictransformation of tissues in culture derived from leaf discs orhypocotyl explants. The transformed tissues can be induced to form planttissue structures, which can be regenerated into whole plants.

[0080] The transformation technique of the present invention is onewhich makes use of the Ti plasmid of A. tumefaciens. In using an A.tumefaciens culture as a transformation vehicle, it is most advantageousto use a non-oncogenic strain of the Agrobacterium as the vector carrierso that normal non-oncogenic differentiation of the transformed tissueis possible. To be effective once introduced into plant cells, thechimeric construction including a foreign gene of interest must containa promoter which is effective in plant cells to cause transcription ofthe gene of interest and a polyadenylation sequence or transcriptioncontrol sequence also recognized in plant cells. Promoters known to beeffective in plant cells include the nopaline synthase promoter,isolated from the T-DNA of Agrobacterium, and the cauliflower mosaicvirus 35S promoter. Other suitable promoters are known in the art. It isalso preferred that the vector which harbors the foreign gene ofinterest also contain therein one or more selectable marker genes sothat the transformed cells can be selected from non-transformed cells inculture. In many applications, preferred marker genes include antibioticresistance genes so that the appropriate antibiotic can be used tosegregate and select for transformed cells from among cells which arenot transformed.

[0081] The details of the construction of the vectors containing suchforeign genes of interest are known to those skilled in the art of plantgenetic engineering and do not differ in kind from those practices whichhave previously been demonstrated to be effective in tobacco, petuniaand other model plant species. The foreign gene should obviously beselected as a marker gene (Jefferson et al. (1987) EMBO J. 6:3901-3907)or to accomplish some desirable effect in plant cells. This effect maybe growth promotion, disease resistance, a change in plant morphology orplant product quality, or any other change which can be accomplished bygenetic manipulation. The chimeric gene construction can code for theexpression of one or more exogenous proteins, or can cause thetranscription of negative strand RNAs to control or inhibit either adisease process or an undesirable endogenous plant function.

[0082] To initiate the transformation and regeneration process for planttissues, it is necessary to first surface sterilize tissues to preventinadvertent contamination of the resulting culture. If the tissues areseeds, the seeds are then allowed to germinate on an appropriategerminating medium containing a fungicide. Four to ten days aftergermination the hypocotyl portion of the immature plant is removed andsectioned into small segments averaging approximately 0.5 centimetersapiece. The hypocotyl explants are allowed to stabilize and remainviable in a liquid or agar plant tissue culture medium.

[0083] Once the tissues have stabilized, they can promptly be inoculatedwith a suspension culture of transformation competent non-oncogenicAgrobacterium. The inoculation process is allowed to proceed for a shortperiod, e.g., two days, at room temperature, i.e., 24° C.

[0084] At the end of the inoculation time period, the remaining treatedtissues can be transferred to a selective agar medium, which containsone or more antibiotics toxic to Agrobacterium but not to plant tissues,at a concentration sufficient to kill any Agrobacterium remaining in theculture. Suitable antibiotics for use in such a medium includecarbenicillin, cefotaxime, etc. as the bactericide for Agrobacterium andkanamycin as the selective antibiotic for transformed plant tissues.

[0085] The tissues are now cultivated on a tissue culture medium which,in addition to its normal components, contains a selection agent. Theselection agent, exemplified herein by kanamycin, is toxic tonon-transformed cells but not to transformed cells which haveincorporated genetic resistance to the selection agent and areexpressing that resistance. A suitable tissue culture medium is the MSmedium to which are added an auxinic analogue of the invention and acytokinin, with or without antibiotics. The surviving transformedtissues are transferred to a secondary medium to induce tissueregeneration. The surviving transformed tissue will thus continue to beregenerated into a whole plant through the regeneration technique of thepresent invention or through any other alternative plant regenerationprotocols.

[0086] The precise amount of growth affecting compositions employed inthe practice of the present invention will depend upon the type ofresponse desired, the formulation used and the type of plant treated.The invention contemplates the use of a ratio of cytokinin concentrationto auxin concentration of between approximately 50.0 and 0.001, andpreferably between approximately 5.0 and 0.05, and more preferablybetween approximately 2.0 and 0.25.

[0087] The chemical compounds employed as active components of thegrowth enhancing compositions of the present invention may be preparedin accordance with processes well known in the prior art or may beobtained commercially from readily available sources.

[0088] The IAA analogues of the invention are useful in making a plantless susceptible to the toxicities of antibiotics. Such IAA analoguesare also useful in enabling plants to overcome stress, e.g.,environmental stress, physical stress, chemical stress, pollution,contamination, drought, light, and the like.

[0089] The present compositions may be applied at any developmentalstage of the plant species to obtain plant hormone or maintenanceeffects throughout maturity and to expedite regrowth in damaged tissuesduring early developmental stages, depending upon the concentrationused, the formulation employed and the type of plant species treated.

[0090] The compositions of the present invention are preferably used inconjunction with specific auxiliary nutrients or other plant growthregulators in precise proportions to achieve a particular synergistic,growth enhancing response in various type of plants. The presentcompositions may additionally be used in association with fungicides toincrease the disease resistance of various plants, making the planttissue resistant to invasion by pathogens by influencing the enzyme andplant processes which regulate natural disease immunity. While thepresent compositions possess essentially no phytotoxic activity of theirown, they may sometimes be used in conjunction with herbicides tostimulate the growth of unwanted plants in order to make such plantsmore susceptible to a herbicide. However, it is preferred to regard theresults achieved in the practice of the present invention as growthenhancing responses in agricultural and horticultural crops, as well asperennial and annual household plants species.

[0091] The following examples are illustrative of the wide range ofplant growth responses that can be realized by application of apreferred composition of the present invention to various plant species.Nevertheless, there is no intention that the invention be limited tothese optimum ratios of active components since workers in the art willfind the compositions of the invention set forth hereinabove to beeffective growth enhancers. Also, it should readily occur to one skilledin the art that the recognition of improved results using thecompositions according to the present invention in connection with otherplants, seeds, fruits and vegetables not specifically illustrated hereinis readily within the capabilities of one skilled in the art. Thefollowing examples serve to illustrate the utility of the inventionwithout limiting its scope.

EXAMPLES Example 1 Chemical Synthesis of an IAA Auxinic Analogue

[0092] (A) Synthesis of an Halogenated IAA

[0093] The synthesis of monohalogenated IAA compounds is based primarilyon the Fischer indole synthesis method as put forth by J. March inAdvanced Organic Chemistry (1985) J. Wiley and Son, p. 1032-1033. Thismethod depends upon Fischer ring closure of the proper 3-formylpropionicacid phenyl hydrazone. The general availability of substitutedphenylhydrazine has made this method attractive for the preparation ofthe indole-3-acetic acids substituted in the benzene ring (Fox andBullock (1951) J. Am. Chem. Soc. 73:2756 and Hatano et al. (1987)Experientia 43:1237).

[0094] Alternatively, a large variety of substituted anilines areavailable and can be used as starting materials to synthesizecorresponding substituted phenylhydrazines which, in turn, can beconverted to substituted IAA compounds. Substituted anilines can beconverted to corresponding substituted phenylhydrazines by the method ofRobinson (1957) Can. J. Chem. 35:1570.

[0095] Use of the 2- and 4-halogen substituted phenylhydrazine with the3-formylpropionic acid gives rise to a 2- and 4-halogen substitutedphenylhydrazone which cyclizes to the corresponding 7- and 5-halo-IAA,respectively. Preparation of the 3-halogen substituted phenylhydrazonecyclizes to form the 4- and 6-halo-IAA isomers which can be separatedusing C₁₈ reverse phase HPLC by elution with water:methanol:acetic acid.The 4-halo-IAA is eluted out in advance of the 6-halo-IAA. A 2-halo-IAAcompound can be synthesized by the procedure described by Porter andThimann (1965) 4:229-243. Each isomeric product can be identified usingthin layer chromatography, mass spectroscopy and nuclear magneticresonance.

[0096] Both a 4-halo-IAA and a 6-halo-IAA compound can be synthesizedindividually by the method of Majima and Hoshino (1925) Ber 58:2042 asdescribed by Fox and Bullock (1951) J. Am. chem. Soc. 73:2756. The 4-and 6-chloroindolylmagnesium iodide complexes are condensed withchloroacetonitrile and the resulting nitrites are hydrolyzed to thecorresponding indole-3-acetic acids.

[0097] The protocol for the synthesis of a monosubstituted halogen-IAAcomprises the following steps. A substituted phenylhydrazine HCl (0.05mole) is dissolved in 30% acetic acid, pH 4.0, and is added to 0.1 mole3-formylpropionic acid solution that is freshly-prepared (See below).After cooling, the precipitate is collected and dissolved in 75 mlpyridine. To the solution, 100 ml of concentrated HCl and 25 ml of 85%H₃PO₄ are added and the resultant solution is refluxed for 10 hours inthe dark under N₂.

[0098] The reaction mixture is diluted with 600 ml H₂O, filtered and thefiltrate is extracted with ether several times. The ether fractions arepooled and washed with water. The indole acetic acid is extracted backinto 0.5 M NaOH (200 ml), boiled and precipitated with concentrated HCl(pH 1.0). Soapy tars are decanted or filtered off. The substituted IAAis recrystallized twice from H₂O, H₂O/ethanol, toluene or ethylacetate/hexane.

[0099] 3-formylpropionic acid is freshly prepared by adding 200 ml offresh 1 M NaOCl solution to 24.9 g (0.20 mole) of glutamic acid in 400ml of 0.5 N NaOH solution, stirring until it gives a negative test withstarch-iodide paper and is then acidified by adding 70 ml of 3N HCl.

[0100] Multi-halogenated IAA compounds can be synthesized according tothe guidelines provided by the methods of Engvild (1977) Acta Chem.Scand. B31:338 (e.g., 4,6-, 4,7-, 5,7-, 6,7-dichloro-IAAs), or themethod of Baldi et al. (1985) J. Label. Compd. Radiopharm. 22:279 (e.g.,5,6-, 4,5-dichloro-IAAs) or the method of Hatano et al. (1987)Experientia 43:1237-1239 (e.g., 5,6-, 6,7-, 4,5-, 4,6-, 5,7-,4,7-dichloro-IAA), etc.

[0101] (B) Synthesis of an Alkyl-, Alkoxy-, Acyl-, Acylamido- andAcyloxy-IAA

[0102] The procedure outlined above (Example 1(A)) is used to preparemonosubstituted IAA compounds having an alkyl-, alkoxy-, acyl-,acylamido- or acyloxy-substituted group at the 2, 4, 5, 6 or 7 position.The synthetic reaction is carried out using the appropriate substitutedphenylhydrazine in order to obtain the desired substituted IAA compound.Where necessary, a reactive substituent group, e.g., acyl-, acylamido-,acyloxy-, etc., may be protected during the synthetic process afterwhich it is deprotected.

[0103] Multi-substituted IAA analogues have 2 or more differentsubstituents selected from halo-, alkyl-, alkoxy-, acyl-, acylamido-,acyloxy-substituent groups can be synthesized using synthetic techniqueswell known in the art [Vasil and Vasil (1994) in Plant Cell and TissueCulture (Vasil and Thorpe, eds.), Kluwer Academic Publishers, Dordrech,Netherlands; Chee (1995) Plant Cell Reports 14:753-757; Bums andSchwartz (1996) Plant Cell Reports 15:405-408; Mihaljevic et al. (1996)Plant Cell Reports 15:610-614; Schopke et al. (1996) NatureBiotechnology 14:731). For example, IAA compounds comprisingcombinations of alkyl-, halo- and acyl-substituent groups (e.g.,2-methyl-5,7-dichloro-IAA, 2-COOH-5-methyl-IAA, 2-COOH-7-chloro-IAA,etc.) can be prepared according to Fox and Bullock (1951) J. Am. Chem.Soc. 73:2756-2759; Hoffman et al. (1952) J. Biol. Chem. 196:437 andEngvild (1977) Acta Chem. Scand. B31:338-344, etc.

Example 2 Evaluation of Auxin Activity in Non-Transgenic Plants

[0104] (a) Tobacco

[0105] Seeds of Nicotiana tobaccum Xanthi are provided by Dr. JamesSaunders (USDA, Beltsville, Md.). Young tobacco leaves are removed andcut into small pieces. The explants are incubated on (1) MS completemedium (obtained from Life Technologies, Inc., Gaithersburg, Md.)containing different concentrations of auxin related compounds only or(2) MS complete medium containing different concentrations ofauxin-related compounds and 0.5 mg/l benzylaminopurine (BAP) using an 18h light/6 h dark cycle until the formation of green calli and shoots.

[0106] (b) Tomato

[0107] Tomato seeds of variety “Moneymaker” are provided by Dr. JamesSaunders (USDA, Beltsville, Md). Young tomato leaves are removed and cutinto small pieces. Explants are incubated in MS complete medium withauxin in the presence or absence of cytokinin as described in section(a) above for tobacco.

[0108] (c) Potato

[0109] Potato seeds of variety “Kent V. F.” are provided by Dr. JamesSaunders (USDA, Beltsville, Md). Young potato stems are removed and cutinto small pieces. Explants are incubated in MS complete medium withauxin in the presence or absence of cytokinin as described in section(a) above for tobacco.

[0110] (d) Arabidopsis thaliana

[0111] Seeds of Arabidopsis thaliana ecotypes Columbia and Landersbergereta are provided by Dr. Keith Davis (The Ohio State University,Columbus). Tissues of hypocotyl are removed from 10-day-old seedlings,transferred to MS complete medium containing (1) differentconcentrations of IAA or IAA analogue with different concentrations ofN⁶.(Δ₂Isopentenyl) adenine (2iP) and (2) different concentrations of IAAanalogue with different concentrations of 2iP, BAP, or kinetin. Theexplants are incubated at 23° C. using an 18 h light/6 h dark cycleuntil the formation of green calli and shoots.

[0112] (e) Rice

[0113] Seeds of Oryza sativa cv. Orion are kindly provided by Dr. JamesSaunders (USDA, Beltsville, Md). For rice embryogenic callus formation,the surfaces of the seeds are sterilized as follows: mature seeds aresoaked in 0.5% detergent with shaking for 1 h, transferred to a solutioncontaining 20% bleach and 0.1% Tween20® and vacuumed with shaking. Theseeds are then rinsed with sterilized distilled water three times. Atthis point the seeds are transferred onto a MS complete mediumcontaining different concentrations of BAP and different concentrationsof IAA analogue, incubated in the dark at 25° C. for 1 month, and thenincubated at 25° C. using an 18 h light/6 h dark cycle until theformation of green calli and shoots.

[0114] (f) Cassava

[0115] Stems or third young leaves were removed from the one month oldCassava plants and cut into small pieces. The explants were incubated onMS complete medium containing different concentrations of auxin relatedcompounds and 0.5 mg/l benzylaminopurine (BAP). The explants wereincubated at 23° C. using an 18 hour light/six hour dark cycle until theformation of green calli and shoots.

[0116] The regeneration of plant tissues using tissue culture depends onplant hormones such as 11O auxin and cytokinin. It is known that thepresence of an auxin in plant tissue cultured on Murashige and Skoog(MS) medium (Murashige, T. and F. Skoog (1962) Physiol. Plant15:473-497) stimulates the formation of root structure whereas theformation of callus is observed when not only the auxin but also acytokinin complement the MS nutrient medium. Therefore, evaluation of atest compound as a potential new auxin is performed by incubatingtobacco leaf discs in (a) the MS complete medium containing differentconcentrations of auxin only and (b) the MS complete medium containingdifferent ratios of cytokinin to auxin concentrations (cytokinin/auxin).

[0117] In a particular embodiment of mono-substituted IAA analogues,fifteen halogenated IAA compounds are chemically synthesized and testedfor plant growth regulatory activity. As shown in FIG. 2, the followingcompounds are tested for auxin activity: 2-iodo-IAA, 4-iodo-IAA,5-iodo-IAA, 6-iodo-1,7-iodo-IAA, 2-bromo-IAA, 4-bromo-IAA, 5-bromo-IAA,6-bromo-IAA, 7-bromo-IAA, 2-fluoro-IAA, 4-fluoro-IAA, 5-fluoro-IAA,6-fluoro-IAA and 7-fluoro-IAA. The biological activity of each compoundis compared to that of IAA. Different concentrations of each compoundare tested for the ability to stimulate root, shoot and callus formationfrom tobacco and tomato leaf discs and potato stems. The ability tostimulate root formation is evaluated for each compound in the presenceof different concentration ratios of cytokinin (e.g., BAP) to testcompound.

[0118] In another embodiment of mono-substituted IAA analogues, alkylsubstituted IAA compounds are chemically synthesized and tested forplant growth regulatory activity. Shown in FIG. 3 are ninemonosubstituted IAA compounds having an alkyl substituent group atposition 4. The present invention also contemplates alkyl-IAA compoundshaving alkyl substituents with 1-10 carbon atoms and having the same ordifferent alkyl substituent groups at position 2, 5, 6 or 7. Thebiological activity of each compound is compared to that of IAA.Different concentrations of each compound are tested for the ability tostimulate root, shoot and callus formation from tobacco and tomato andtomato leaf discs and potato stems. The ability to stimulate root, shootand callus formation is evaluated for each compound in the presence ofdifferent concentration ratios of cytokinin (e.g., BAP) to testcompound.

[0119] In another embodiment of mono-substituted IAA analogues,alkoxy-substituted IAA compounds are chemically synthesized and testedfor plant growth regulatory activity. Shown in FIG. 4 are ninemonosubstituted IAA compounds having an alkoxy substituted group atposition 4. The present invention also contemplates alkoxy-IAA compoundshaving alkoxy substituent groups with 1-10 carbon atoms and having thesame or different alkoxy substituent groups at position 2, 5, 6 or 7.The biological activity of each compound is compared to that of IAA.Different concentrations of each compound are tested for the ability tostimulate root, shoot and callus formation from tobacco and tomato leafdiscs and potato stems. The ability to stimulate root shoot and callusformation is evaluated for each compound in the presence of differentconcentration ratios of cytokinin (e.g., BAP) to test compound.

[0120] In another embodiment of mono-substituted IAA analogues, acylsubstituted IAA compounds are chemically synthesized and tested forplant growth regulatory activity. Shown in FIG. 5 are ninemonosubstituted IAA compounds having an acyl substituent group atposition 4. The present invention also contemplated acyl-IAA compoundshaving acyl substituents with 1-10 carbon atoms and having the same ordifferent acyl substituent groups at position 2, 5, 6 or 7. Thebiological activity of each compound is compared to that of IAA.Different concentrations of each compound are tested for the ability tostimulate root, shoot and callus formation from tobacco and tomato leafdiscs and potato stems. The ability to stimulate root formation isevaluated for each compound in the presence of different concentrationratios of cytokinin (e.g., BAP) to test compound.

[0121] In another embodiment of mono-substituted IAA analogues,acylamido substituted IAA compounds are chemically synthesized andtested for plant growth regulatory activity. Shown in FIG. 6 are ninemonosubstituted IAA compounds having an acylamido substituent group atposition 4. The present invention also contemplated acylamido-IAAcompounds having acylamido substituents with 1-10 carbon atoms andhaving the same acylamido substituent groups at position 2, 5, 6 or 7.The biological activity of each compound is compared to that of IAA.Different concentrations of each compound are tested for the ability tostimulate root, shoot and callus formation from tobacco and tomato leafdiscs and potato stems. The ability to stimulate root formation isevaluated for each compound in the presence of different concentrationratios of cytokinin (e.g., BAP) to test compound.

[0122] In another embodiment of mono-substituted IAA analogues, acyloxysubstituted IAA compounds are chemically synthesized and tested forplant growth regulatory activity. Shown in FIG. 7 are ninemonosubstituted IAA compounds having an acyloxy substituent group atposition 4. The present invention also contemplated acyloxy-IAAcompounds having acyloxy substituents with 1-10 carbon atoms and havethe same or different acyloxy substituent groups at position 2, 5, 6 or7. The biological activity of each compound is compared to that of IAA.Different concentrations of each compound are tested for the ability tostimulate root, shoot and callus formation from tobacco and tomato leafdiscs and potato stems. The ability to stimulate root formation isevaluated for each compound in the presence of different concentrationratios of cytokinin (e.g., BAP) to test compound.

[0123] In particular embodiments of multi-substituted IAA analogues,di-, tri-, tetra- or penta-substituted IAA compounds, comprising betweentwo and five substituent groups that are individually selected from thegroup consisting of a halo-, an alkyl-, an alkoxy-, an acyl-, anacylamido- and an acyloxy-substituent group in at least two of thepositions corresponding to the 2, 4, 5, 6 or 7 position in the IAAchemical structure of FIG. 1, are synthesized and tested for plantgrowth regulatory activity. In these multi-substituted IAA compounds, ahalo-substituent group includes, but is not limited to, an iodo-, abromo-, a fluoro- and a chloro-group; an alkyl-substituent groupincludes, but is not limited to, a linear, branched or cyclic alkylgroup, R, having 1-10 carbon atoms; an alkoxy-substituent includes, butis not limited to, a linear, branched or cyclic alkoxy group, RO—,having 1-10 carbon atoms; an acyl-substituent group includes, but is notlimited to, a linear, branched or cyclic acyl group, R—C(O)—, having1-10 carbon atoms; an acylamido-substituent group includes, but is notlimited to, a linear, branched or cyclic acylamido-group, R—C(O)—NH—,having 1-10 carbon atoms; and an acyloxy-substituent group includes, butis not limited to, a linear, branched or cyclic acyloxy-group,R—C(O)—O—, having 1-10 carbon atoms.

[0124] For each multi-substituted IAA, its auxinic activity is testedand compared to that of IAA. Different concentrations of each compoundare tested for the ability to stimulate root, shoot and callus formationfrom tobacco and tomato leaf discs and potato stems. The ability tostimulate root formation is evaluated for each compound in the presenceof different concentration ratios of cytokinin (e.g., BAP) to testcompound.

Example 3 The Use of IAA Auxinic Analogues for Stimulating theRegeneration of Transgenic Plants

[0125] (a) Tobacco

[0126] Plant transformation is carried out according to theAgrobacterium-mediated transformation procedure essentially as describedby Lin et al. [(1994) Focus 16:72-77)].

[0127] For tobacco, the leaf discs are incubated with 10¹⁰ cells/mlAgrobacterium tumefaciens LBA4404 cells, containing pBI121 harboring theGUS reporter gene, in MS complete medium with 0.5 mg/l2-(N-morpholino)ethanesulfonic acid (MES) for 10 min, transferred tosolid MS complete medium, and incubated for 2 days at 25° C., using an18 h light/6 h dark cycle for cocultivation. After cocultivation, theexplants are transferred to MS media containing different ratios ofBAP/IAA or BAP/IAA analogue, 100 mg/l kanamycin and 500 mg/lcarbenicillin and incubated at 25° C. using an 18 h light/6 h dark cyclefor shoot formation.

[0128] (b) Tomato

[0129] Plant transformation is carried out according to theAgrobacterium-mediated transformation procedure essentially as describedby Lin et al. (1994) Focus 16:72-77. Incubation conditions are asdescribed above in section (a) for tobacco.

[0130] (c) Potato

[0131] Plant transformation in potato stems is carried out as describedabove in section (a) for tobacco.

[0132] (d) Arabidopsis thaliana

[0133] For Arabidopsis thaliana, tissues of hypocotyl are removed from10-day-old seedlings and preincubated in MS complete medium containing0.5 mg/l 2,4-D and 0.5 mg/l kinetin for three days. The explants areimmersed in 10⁹ cells/ml of Agrobacterium tumefaciens LBA4404 containingpBI121 for 20 min, and transferred to solid MS medium containing 500mg/l carbenicillin, 50 mg/l kanamycin, and various ratios of IAAanalogue/2iP or IAA/2iP for callus and shoot formation. Arabidopsisexplants were incubated at 25° C., using an 18 h light/6 h dark cycle.

[0134] (e) Rice.

[0135] The transformation of monocotyledonous plants is carried outaccording to art-known methods as described by Wu, “Methods forTransforming Plant Cells,” in Plant Biotechnology (1989), Kung andArntzen, Eds., Butterworth Publishers, Stoneham, Mass. It is preferredthat transformation of monocots such as rice and wheat be performed bythe particle bombardment method as described in Wang et al. (1988) PlantMol. Biol. 11:433-439. The regeneration of transformed monocots isperformed according to known procedures (Vasil, Biotechnology (1988)5:387-402) as described in Example 5.

[0136] For example, rice (Oryza sativa) is transformed using theparticle bombardment method of Wang et al. (supra) or theAgrobacterium-mediating technique of Hiel et al. (1994) Plant Journal6:271 or, alternatively, using the electroporation method as describedby Dekeyser et al. (1990) Plant Cell 2:591-602. Regeneration oftransformed rice is performed according to Abdullah et al. (1986)Bio/Technology 4:1087-1909 or, alternatively, according to Raineri etal. (1990) Bio/Technology 8:33-38.

[0137] In a further example, maize is transformed and regeneratedaccording to the procedures of Rhodes et al. (1988) Bio/Technology6:56-60 and (1988) Science 240:204-207.

[0138] In all cases, an IAA auxinic analogue is used as the auxin tostimulate plant growth in accordance with the invention. Where required,one or more additional plant growth regulators may be added to the IAAanalogue-comprising plant growth compositions.

[0139] (f) Cassava.

[0140] Stems or leaves preferably the first, second or third leaf, andmore preferably the bottom quarter of the leaf closest to the petiole)from one-month-old cassava plants were cut into small pieces. ForeignDNA was inserted by techniques known in the art, e.g., A. tumifacienstechnique [Lin et al. (1994) supra] or with the microbombardmenttechnique [Schopke et al. (1996) Nature Biotechnology 14:731].Transgenic cassava tissue was exposed to IAA analogues of the inventionand examined for regeneration of cassava tissues.

Example 4 Screening of Substituted IAA Compounds for Auxinic Activity

[0141] (a) Non-Transgenic Plants as documented in FIGS. 8-10 and Tables1, 2 and 4.

[0142] Seeds of Nicotiana tobaccum Xanthi, potato and tomato were kindlyprovided by Dr. James Saunders (USDA, Beltsville). Cassava plants wereprovided by Dr. Dick Sayer (The Ohio State University, Columbus, Ohio).

[0143] Young tobacco leaves, potato stems, tomato leaves and cassavaleaves and stems from one-month-old plants were removed and cut intosmall pieces. The explants were incubated on (1) the MS complete mediumcontaining different concentrations of auxin related compounds only (2)the MS complete medium containing different concentrations ofauxin-related compounds and 0.5 mg/l benzylaminopurine (BAP) using an 18hour light/6 hour dark cycle until the formation of green calli, shootsor roots.

[0144] The results of root, shoot and callus formations were shown intomato (FIG. 8), potato (FIG. 9) and tobacco (FIG. 10) and cassava (FIG.11) incubated in MS complete medium comprising 6-BrIAA only or 6-BrIAAwith BAP. The results of screening different IAA analogues for auxinicactivities were presented in Table 1 for MS complete medium with IAAanalogue only, in Table 2 for MS complete medium with IAA analogue andBAP, and in Table 4 for regeneration of cassava in MS complete mediumwith different IAA analogues and BAP.

[0145] (b) Transgenic Plants as Documented in Table 3.

[0146] Agrobacterium-mediated plant transformation was performed asdescribed by Lin et al. (1994) Focus 16:72-77. For example:

[0147] (i) Tobacco

[0148] In tobacco, the leaf discs were incubated with 10⁹ cells/ml ofAgrobacterium tumefaciens LBA4404 cells containing pBI121 in MS completemedium with 0.5 mg/l MES for 10 minutes, transferred to solid MScomplete medium, and incubated for two days at 25° C., using an 18 hourlight/6 hour dark cycle for cocultivation. After cocultivation, theexplants were transferred to the MS medium containing a different ratioof BAP/IAA or BAP/IAA analogue, 100 mg/l kanamycin and 500 mg/lcarbenicillin and incubated at 25° C. using an 18 hour light/6 hour darkcycle for shoot formation.

[0149] (ii) Potato

[0150] In another example, potato leaf discs were transformed with theAgrobacterium-mediation technique as described above for tobacco. Thepotato explants were then subjected to incubation with IAA analogues inthe absence and presence of cytokinin to screen for auxinic activities.

[0151] (iii) Cassava

[0152] Stems or third young leaves were removed from the one-month-oldCassava plants and cut into small pieces. The explants were incubatedwith 10¹⁰ A. tumefaciens cells containing pBI121 for 20 minutes in MScomplete liquid medium, transferred to MS complete solid medium andincubated for two days. After two days' incubation, the explants weretransferred to MS complete medium with 500 mg/l carbenicillin, 50 mg/lkanamycin, 0.5 mg/l BAP, and different concentrations of auxin relatedcompounds. The explants were incubated at 23° C. using an 18 hourlight/six hour dark cycle until the formation of green calli and shoots.

Example 5 Alleviation of Stress with Auxin and Auxin Derivatives

[0153] (a) Attenuation of Chemical Toxicity.

[0154] Potato stems were excised as described above and incubated in theMS complete medium (a) with 25, 50 and 100 mg/l kanamycin and eitherwith/without 2 mg NAA or auxin derivatives or with/without 0.5 mg/l BAP(a ctyokinin). After one month incubation, potato stems incubated in 50mg or 100 mg/l kanamycin with/without BAP only showed necrosis of stemwhich indicted the toxicity of kanamycin. However, potato stemsincubated in the MS medium containing 2 mg/l NAA and 100 mg/kanamycin,became expanded (FIG. 12). In some cases, the presence of auxin or auxinderivatives showed the formation of calli in some of the potato stems.

[0155] In order to confirm that the auxin has the ability to restore theviability of antibiotic treated potato stems, the stems incubated forone month with 50 mg/l kanamycin and 0.5 mg/l BAP were transferred intoMS complete medium containing 2 mg/l NAA with/without 100 mg/lkanamycin, or MS medium containing 2 mg/l NAA, 0.5 mg/l BAP, andwith/without 100 mg/l kanamycin. The formation of calli on the potatostems was observed when the potato stems were transferred to the MSmedium containing auxin or auxin derivative with/without BAP. However,all the potato stems were dead after continued incubation without theaddition of auxin or auxin derivatives (FIG. 13).

[0156] Similar antibiotics and auxin interactions were observed in themedium containing 2, 4-D, 5-bromo-IAA and IAA. In addition, differentantibiotics such as hygromycin and chloramphenicol showed the sameinteractions with auxin treated potato stems and tobacco leaves.

[0157] B. Alleviation of Physical Damage with Auxin and AuxinDerivatives.

[0158] Tobacco leaves were removed from one month old plants and cutinto small pieces (0.5 c×0.25 cm). These tissues were incubated in theMS complete medium in 0.5 mg/l BAP overnight and bombarded withmicroprojectiles (1300 psi) (helium source, obtained from BioRad). Afterbombardment, the tobacco explants were incubated in (1) MS completemedium with 0.5 mg/l BAP, and (2) MS complete medium with 0.5 mg/l BAPand 2 mg/l NAA for three days before transferring to MS complete mediumwith 0.5 mg/l BAP. Significant improvement of the regeneration oftobacco shoots were shown in those explants incubated in MS mediumcontaining NAA and BAP compared to those shoots incubated in MS mediumwith BAP only. Moreover, similar results were shown when the tobaccoexplants were bombarded with pBI121 and incubated with 0.5 mg/l BAP andwith/without 2 mg NAA. The transformed tissues were selected byincubation of the explants in MS complete medium with 0.5 mg/l BAP and100 mg/l kanamycin. All these results demonstrated that auxin were ableto alleviate the physical stress such as bombardment and restore theregeneration of plant tissues.

[0159] All publications, patent applications and patents cited hereinare incorporated by reference in the same extent as if each individualpublication, patent application or patent was specifically andindividually indicated to be incorporated by reference.

We claim:
 1. A method for generation of callus, shoots, or roots from a plant cell or tissue which comprises the step of contacting said plant cell or plant tissue with an amount of an auxinic compound effective for generation of callus, shoots or roots and having the formula:

or salts, esters or amides thereof, wherein R₁-R₅ are independently selected from the group consisting of a hydrogen, a halogen, an alkyl-group, an alkoxy-group, an acyl-group, an acyloxy-group, and an acylamido-group, but excluding indole-3-acetic acid and 5-bromoindole-3-acetic acid.
 2. The method of claim 1 wherein said alkyl-group, said alkoxy-group, said acyl-group, said acyloxy-group and said acylamido-group have 1-10 carbon atoms.
 3. The method of claim 1 wherein said auxinic compound is a monosubstituted indole-3-acetic acid or salt, ester or amide thereof.
 4. The method of claim 3 wherein in said auxinic compound one of R₁-R₅ is a bromine, a fluorine or an iodine.
 5. The method of claim 3 wherein in said auxinic compound one of R₁-R₅ is a chlorine.
 6. The method of claim 1 wherein said auxinic compound is a disubstituted indole-3-acetic acid.
 7. The method of claim 6 wherein in said auxinic compound at least one of R₁-R₅ is a bromine, or a fluorine.
 8. The method of claim 6 wherein in said auxinic compound at least one of R₁-R₅ is a chlorine.
 9. The method of claim 1 wherein said auxinic compound is 2-fluoroindole-3-acetic acid, 4-fluoroindole-3-acetic acid, 6-fluoroindole-3-acetic acid, 7-fluoroindole-3-acetic acid, 2-bromoindole-3-acetic acid, 4-bromoindole-3-acetic acid, 6-bromoindole-3-acetic acid, 7-bromoindole-3-acetic acid, 2-iodoindole-3-acetic acid, 4-iodoindole-3-acetic acid, 5-iodoindole-3-acetic acid, 6-iodoindole-3-acetic acid, 7-iodoindole-3-acetic acid or salts, esters or amides thereof.
 10. The method of claim 1 wherein said auxinic compound is 2-fluoroindole-3-acetic acid, 4-fluoroindole-3-acetic acid, 6-fluoroindole-3-acetic acid, 7-fluoroindole-3-acetic acid or salts, esters or amides thereof.
 11. The method of claim 1 wherein said auxinic compound is 6-fluoroindole-3-acetic acid, 7-fluoroindole-3-acetic acid or salts, esters or amides thereof.
 12. The method of claim 1 wherein said auxinic compound is 2-bromoindole-3-acetic acid, 4-bromoindole-3-acetic acid, 6-bromoindole-3-acetic acid, 7-bromoindole-3-acetic acid or salts, esters or amides thereof.
 13. The method of claim 1 wherein said auxinic compound is 4-bromoindole-3-acetic acid, 6-bromoindole-3-acetic acid, 7-bromoindole-3-acetic acid or salts, esters or amides thereof.
 14. The method of claim 1 wherein said auxinic compound is 6-bromoindole-3-acetic acid, 7-bromoindole-3-acetic acid or salts, esters or amides thereof.
 15. The method of claim 1 wherein said auxinic compound is 2-iodoindole-3-acetic acid, 4-iodoindole-3-acetic acid, 5-iodoindole-3-acetic acid, 6-iodoindole-3-acetic acid, 7-iodoindole-3-acetic acid or salts, esters or amides thereof.
 16. The method of claim 1 wherein in said auxinic compound one of R₁-R₅ is an alkyl-group and the remaining R₁-R₅ groups are hydrogens.
 17. The method of claim 16 wherein in said auxinic compound one of R₁-R₅ is an alkyl-group having from one to four carbon atoms.
 18. The method of claim 17 wherein one of said R₁-R₅ groups is an ethyl group.
 19. The method of claim 18 wherein said auxinic compound is 7-ethylindole-3-acetic acid, 5-ethylindole-3-acetic acid, or salts, esters or amides thereof.
 20. The method of claim 1 wherein in said auxinic compound one of R₁-R₅ is an alkoxy-group and the remaining R₁-R₅ groups are hydrogens.
 21. The method of claim 20 wherein in said auxinic compound one of R₁-R₅ is an alkoxy-group having from one to four carbon atoms and the remaining R₁-R₅ groups are hydrogens.
 22. The method of claim 21 wherein one of said R₁-R₅ groups is a methoxy group.
 23. The method of claim 22 wherein said auxinic compound is 5-methoxyindole-3-acetic acid or salts, esters or amides thereof.
 24. The method of claim 1 wherein said plant cell or plant tissue comprises foreign DNA.
 25. The method of claim 24 wherein said auxinic compound is 5-fluoroindole-3-acetic acid, 7-fluoroindole-3-acetic acid, 7-bromoindole-3-acetic acid, 5-chloroindole-3-acetic acid, 5-ethylindole-3-acetic acid, 7-ethylindole-3-acetic acid or salts, esters or amides thereof.
 26. The method of claim 1 wherein said plant cell or tissue is that of a hard-to-regenerate plant.
 27. The method of claim 26 wherein said plant cell or tissue is that of a woody plant or a monocotyledonous plant.
 28. The method of claim 26 wherein said plant cell or tissue is a cell or tissue of a cassava plant.
 29. The method of claim 28 wherein in said auxinic compound R₃ or R₅ is a fluorine.
 30. The method of claim 29 wherein said auxinic compound is 5-fluoro-3-acetic acid or salts, esters, or amides thereof.
 31. The method of claim 1 for generation of roots from plant tissue.
 32. The method of claim 31 wherein said auxinic compound is a monosubstituted indole-3-acetic acid or salt, ester or amide thereof.
 33. A method for regeneration of a plant from a plant cell or plant tissue which comprises the steps of: (a) contacting said plant cell or plant tissue with an amount of an auxinic compound effective for regeneration of roots and (b) contacting said plant cell or plant tissue with a combination of said auxinic compound and a cytokinin in a combined amount effective for the regeneration of shoots from said plant cell or plant tissue wherein said auxinic compound has the formula:

 or salts, esters and amides thereof, wherein R₁-R₅ are independently selected from the group consisting of a hydrogen, a halogen, an alkyl-group, an alkoxy-group, an acyl-group, an acyloxy-group, and an acylamido-group, but excluding indole-3-acetic acid and 5-bromoindole-3-acetic acid.
 34. The method of claim 33 wherein said alkyl-group, said alkoxy-group, said acyl-group, said acyloxy-group and said acylamino-group have 1-10 carbon atoms.
 35. The method of claim 33 wherein in said auxinic compound at least one of R₁-R₅ is a halogen.
 36. The method of claim 33 wherein said auxinic compound is 2-fluoroindole-3-acetic acid, 4-fluoroindole-3-acetic acid, 6-fluoroindole-3-acetic acid, 7-fluoroindole-3-acetic acid, 2-bromoindole-3-acetic acid, 4-bromoindole-3-acetic acid, 6-bromoindole-3-acetic acid, 7-bromoindole-3-acetic acid, 2-iodoindole-3-acetic acid, 4-iodoindole-3-acetic acid, 5-iodoindole-3-acetic acid, 6-iodoindole-3-acetic acid, or 7-iodoindole-3-acetic acid.
 37. The method of claim 33 wherein in said auxinic compound at least one of R₁-R₅ is an alkyl group.
 38. The method of claim 33 wherein said plant cell or plant tissue comprises foreign DNA.
 39. The method of claim 33 wherein said plant cell or tissue is of a plant selected from the group of hard to regenerate plants consisting of cassava, woody plants and monocotyledonous plants.
 40. A composition for generation of callus, shoots or roots from a plant cell or tissue which comprises an amount of an auxinic compound effective for generating callus, shoots or roots and having the formula:

or salts, esters and amides thereof, wherein R₁-R₅ are independently selected from the group consisting of a hydrogen, a halogen, an alkyl group, an alkoxy-group, an acyl-group, an acyloxy-group, and an acylamido-group, but excluding indole-3-acetic acid and 5-bromoindole-3-acetic acid.
 41. The composition of claim 40 wherein said auxinic compound, said alkyl-group, said alkoxy-group, said acyl-group, said acyloxy-group and an acylamido-group have 1-10 carbon atoms.
 42. The composition of claim 40 which is useful for the formation of roots from plant cells or tissue.
 43. The composition of claim 40 which is useful for the formation of shoots from plant cells or tissue.
 44. The composition of claim 40 wherein in said auxinic compound at least one of R₁-R₅ is a halogen.
 45. The composition of claim 40 wherein in said auxinic compound at least one of R₁-R₅ is an alkyl group.
 46. The composition of claim 45 wherein in said auxinic compound R₃ or R₅ is an alkyl group having from one to four carbon atoms and R₁, R₂, and R₄ are hydrogens.
 47. The composition of claim 46 wherein said auxinic compound is 5-ethylindole-3-acetic acid, 7-ethylindole-3-acetic acid or salts, esters, or amides thereof.
 48. The composition of claim 47 wherein said auxinic compound is 2-fluoroindole-3-acetic acid, 4-fluoroindole-3-acetic acid, 6-fluoroindole-3-acetic acid, 7-fluoroindole-3-acetic acid, 2-bromoindole-3-acetic acid, 4-bromoindole-3-acetic acid, 6-bromoindole-3-acetic acid, 7-bromoindole-3-acetic acid, 2-iodoindole-3-acetic acid, 4-iodoindole-3-acetic acid, 5-iodoindole-3-acetic acid, 6-iodoindole-3-acetic acid, 7-iodoindole-3-acetic acid or salts, esters or amides thereof.
 49. The composition of claim 40 wherein said auxinic compound is 2-iodoindole-3-acetic acid, 4-iodoindole-3-acetic acid, 5-iodoindole-3-acetic acid, 6-iodoindole-3-acetic acid, 7-iodoindole-3-acetic acid or salts, esters or amides thereof.
 50. The composition of claim 40 wherein said auxinic compound is 6-bromoindole-3-acetic acid, 7-bromoindole-3-acetic acid or salts, esters or amides thereof.
 51. The composition of claim 40 wherein said at least one of R₁-R₅ is an acyl-group, an acyloxy-group, or an acylamido-group.
 52. The composition of claim 40 further comprising a growth-affecting amount of a cytokinin.
 53. An auxinic compound having the formula:

or salts, esters or amides thereof, wherein R₁-R₅ are independently selected from the group consisting of a hydrogen, a halogen, an alkyl-group, an alkoxy-group, an acyl-group, an acyloxy-group, and an acylamido-group, and wherein at least one of R₁-R₅ is an alkoxy-group, an acyl-group, an acyloxy-group or an acylamido-group with the exception that the compound is not 5-methoxyindole-3-acetic acid.
 54. The compound of claim 53 wherein said alkyl-group, said alkoxy-group, said acyl-group, said acyloxy-group and said acylamido-group have 1-10 carbon atoms.
 55. The compound of claim 54 wherein at least one of R₁-R₅ is an acyl-group, an acyloxy-group or an acylamido-group.
 56. An auxinic compound having the formula:

or salts, esters or amides thereof, wherein R₁-R₅ are independently selected from the group consisting of a hydrogen, a fluorine, a bromine, an iodine, an alkyl group, an alkoxy-group, an acyl-group, an acyloxy-group, and an acylamido-group, wherein at least one of R₁-R₅ is an alkoxy-group, an acyl-group, an acyloxy-group or an acylamido-group and with the exception that the auxinic compound is not 5-bromoindole-3-acetic acid, 7-bromoindole-3-acetic acid, 5-fluoroindole-3-acetic acid, 2-methylindole-3-acetic acid, 5-methylindole-3-acetic acid, or 5-methoxyindole-3-acetic acid.
 57. The auxinic compound of claim 56 which is 2-iodoindole-3-acetic acid, 4-iodoindole-3-acetic acid, 5-iodoindole-3-acetic acid, 6-iodoindole-3-acetic acid, 7-iodoindole-3-acetic acid or salts, esters, or amides thereof.
 58. The auxinic compound of claim 56 which is 5-ethylindole-3-acetic acid, 7-ethylindole-3-acetic acid or salts, esters or amides thereof.
 59. The auxinic compound of claim 56 wherein one of R₃ or R₅ is an ethyl group.
 60. A method for generation of roots from a potato cell or tissue which comprises the step of contacting said potato cell or tissue with an amount of 7-ethyl indole-3-acetic acid effective for generation of roots. 